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Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction
Published on: September 14, 2018
Sortase A-Generated Highly Potent Anti-CD20-MMAE Conjugates for Efficient Elimination of B-Lineage Lymphomas
Liqiang Pan1,2, Wenbin Zhao1, Jun Lai1
1Institute of Drug Metabolism and Drug Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.
Abstract:
Antibody-drug conjugate (ADC) targeting antigens expressed on the surface of tumor cells are an effective approach for delivering drugs into the cells via antigen-mediated endocytosis. One of the well-known tumor antigens, the CD20 of B-lymphocyte, has long been suggested to be noninternalizing epitope, and is thus not considered a desirable target for ADCs. Here, sortase A (srtA)-mediated transpeptidation is used to specifically conjugate triple glycine-modified monomethyl auristatin E (MMAE), a highly toxic antimitotic agent, to anti-CD20 ofatumumab (OFA) equipped with a short C-terminal LPETG (5 amino acids) tag at heavy chain (HL), which generates ADCs that show extremely strong potency in killing CD20 positive cancer cells. One of the srtA-generated ADCs with a cleavable dipeptide linker (valine-citrulline, vc), OFA-HL-vcMMAE, shows IC50 values ranging from 5 pg mL-1 to 4.1 ng mL-1 against CD20+ lymphoma cells. Confocal laser scanning microscopy confirms that OFA-HL-vcMMAE internalization by Ramos cells is significantly improved compared to OFA alone, consistent with the high antitumor activity of the new ADC. OFA-HL-vcMMAE, at 5 mg kg-1 dose, is able to eliminate tumors with mean volume ≈400 mm3 while no obvious drug-related toxicity is observed. The results show that srtA-generated OFA-MMAE conjugate system provides a viable strategy for targeting CD20+ B lineage lymphomas.
Insights
This study developed a novel antibody-drug conjugate (ADC) targeting CD20+ B-lymphoma cells. The new conjugate, OFA-HL-vcMMAE, demonstrates potent tumor cell killing and significant tumor reduction with minimal toxicity.
Area of Science:
- Oncology
- Immunology
- Bioconjugation Chemistry
Background:
- Antibody-drug conjugates (ADCs) offer targeted cancer therapy by delivering cytotoxic drugs to tumor cells.
- The CD20 antigen on B-lymphocytes is a known target, but its non-internalizing nature has limited ADC development.
- Sortase A (SrtA) ligation provides a specific method for antibody-drug conjugation.
Purpose of the Study:
- To develop a potent ADC targeting CD20+ B-lineage lymphomas using SrtA-mediated conjugation.
- To evaluate the efficacy and internalization of the novel ADC in preclinical models.
Main Methods:
- Sortase A (SrtA) transpeptidation was used to conjugate triple glycine-modified monomethyl auristatin E (MMAE) to anti-CD20 ofatumumab (OFA).
- The resulting ADC, OFA-HL-vcMMAE, featured a cleavable valine-citrulline (vc) linker.
- In vitro cytotoxicity assays and confocal microscopy were performed on CD20+ lymphoma cells.
- In vivo studies assessed tumor elimination and toxicity in a mouse model.
Main Results:
- OFA-HL-vcMMAE exhibited potent cytotoxicity against CD20+ lymphoma cells with IC50 values as low as 5 pg/mL.
- Confocal microscopy showed significantly enhanced internalization of OFA-HL-vcMMAE compared to OFA alone.
- A single dose of 5 mg/kg OFA-HL-vcMMAE eradicated established tumors (mean volume ≈400 mm³) without observable toxicity.
Conclusions:
- SrtA-mediated conjugation of OFA with MMAE creates a highly effective ADC for targeting CD20+ B-lineage lymphomas.
- The enhanced internalization and potent antitumor activity validate this approach for CD20-targeting therapies.
- This conjugate system presents a viable strategy for treating CD20-positive B-cell malignancies.
